Intensification of resveratrol cytotoxicity, pro-apoptosis, oxidant potentials in human colorectal carcinoma HCT-116 cells using zein nanoparticles

Resveratrol (RSV), a non-flavonoid stilbene polyphenol, possesses anti-carcinogenic activities against all the major stages of cancer. Zein nanoparticles (ZN NPs) have been utilized successfully in delivery of variant therapeuticals by virtue of their histocompatible nature. The goal of this work was to comparatively explore the antiproliferative, pro-apoptotic and oxidative stress potentials of RSV-ZN NPs versus RSV against human colorectal carcinoma HCT-116 cells. ZN-RSV NPs were developed and assayed for particle size analysis and RSV diffusion. The selected formula obtained 137.6 ± 8.3 nm as mean particle size, 29.4 ± 1.8 mV zeta potential, 92.3 ± 3.6% encapsulation efficiency. IC50 of the selected formula was significantly lower against HCT-116 cells versus Caco-2 cells. Also, significantly enhanced cellular uptake was generated from RSV-ZN NPs versus free RSV. Enhanced apoptosis was concluded due to increased percentage cells in G2-M and pre-G1 phases. The pro-apoptotic potential was explained by caspase-3 and cleaved caspase-3 increased mRNA expression in addition to NF-κB and miRNA125b decreased expression. Biochemically, ZN-RSV NPs induced oxidative stress as demonstrated by enhanced reactive oxygen species (ROS) generation and endothelial nitric oxide synthase (eNOS) isoenzyme increased levels. Conclusively, ZN-RSV NPs obtained cell cycle inhibition supported with augmented cytotoxicity, uptake and oxidative stress markers levels in HCT-116 tumor cells in comparison with free RSV. These results indicated intensified chemopreventive profile of RSV due to effective delivery utilizing ZN nano-dispersion against colorectal carcinoma HCT-116 cells.


Results and discussion
RSV was studies extensively along last decade for obtaining cancer therapeutics promising outcomes in both cancer prevention and treatment. The anticancer molecular mechanism is still not elucidated. Effective formulation of RSV was frequently faced the difficulty of very low oral bioavailability due to reduced water solubility and low photostability. Just less than 1% bioavailability was reported due to severe metabolism in liver and intestines 39 . Pharmaceutical nanotechnological techniques offer successful approach to overcome this obstacle. Nanostructures of RSV are highly recommended to increase water solubility, facilitate biological membranes crossing, ameliorate absorption and stability and enhance pharmacokinetic parameters and bioavailability 40 . This will have significant and promising effect on therapeutical potential of RSV to ameliorate its cytotoxicity and pro-apoptotic activity against HCT-116 cells. The efficiency of nanostructures loaded with RSV will extended to overcome the suggested multiple drug resistance in various tumors and minimize toxicity via enhancing permeation and retention effect of nanostructures 41  www.nature.com/scientificreports/ Characterization of ZN-RSV NPs. RSV Nanocomposites were fabricated in three variant formulae; R1, R2 and R3. Table 1 displayed formulation characteristics for all developed formulae. The most reduced mean particle size was related to formula R1 which was 137.6 ± 8.3 nm. This obtained mean particle size of ZN-RSV NPs is suggested to enhance absorption and residence time inside biological systems 34 . As shown, particle size reduction was directly related to elevation of ZN content through the prepared formulae. The higher particle size of R3 formula could be generated due increased quantity of RSV on nanosphere surface, which could lead to maximized interfacial tension between particle surface and aqueous medium, so consequently particle diameter will be enlarged 42 . Also, R1 formula obtained the highest zeta potential magnitude (29.4 ± 1.8 mV) and the least polydispersity index magnitude (0.19 ± 0.01). Elevated zeta potential value reflects nanostructures dispersion stability as it will withstand aggregations because of charge of ZN surface 35 . R1 formula obtained 92.3% ± 3.6 encapsulation efficiency. The enhanced EE by increasing of in ZN amount in formula could be related to RSV partitioning to the hydrophobic matrix of ZN. The elevated encapsulation efficiency by elevation in ZN amount in formula might be due to integrated partitioning of RSV to ZN hydrophobic matrix which reflects a relation between RSV binding affinity and ZN polymerization degree 43 . The ability of nano-delivery systems to incorporate high percent of payloads was previously reported 26,40,44 . The method utilized for fabrication was adequate in obtaining high encapsulation efficiency values. This could be explained regarding to the natural unfolding of ZN molecules at alkaline pH which offers more reactive sites for maximized crosslinking and also remarkable reduction of void spaces inside ZN nanospheres 35 which will positively contribute to ameliorated bioavailability, delivery and consequently the therapeutical response 45,46 . ZN-RSV NPs formula diffusion is presented in Fig. 1. R1, R2 and R3 formulae displayed sustained RSV diffused profile, which obtained magnitudes of 94.1 ± 3.6%, 90.3 ± 4.5 and 84.4% ± 6.9 after 36 h, respectively. R1 formula was chosen for further investigations. Sustaining RSV diffusion could be attributed to ZN nature hydrophobic structure. ZN generates a delay in the water penetration which could attributed to the measured RSV sustained diffusion 34 . RSV diffusion exhibited biphasic sustained permeation profile. The diffusion displayed initial burst effect related to rapid release of trapped RSV to direction of ZN NPs surfaces. After initial burst, a reduction in diffusion rate was attained as RSV has to penetrate longer path in core matrix to completely release from nanospheres. Also, RSV diffusion rate was affected by water intake rate 42,45,47 . The prolonged release of RSV due to ZN NPs will support the pharmacokinetic potential due to short half-life and reported irregular profile in human after oral dose of RSV by 48-50 that obtained two peak plasma concentration after 1 h and 6 h due to enteric recirculation.
The particles morphology of R1 formula was examined by SEM imaging and showed compactly dispersed spheres with smooth surfaces (Fig. 2). The diameter of scanned spheres was consistent with particle size measured by laser diffraction technique. The noticeable compactness of nano-particulates may be related to freeze drying process 51 .
Serum stability. ZN-RSV NPs colloidal stability was estimated by monitoring the changes in sample mean particle size (Fig. 3). After incubation in FBS, ZN-RSV NPs displayed initial elevation in its mean particle size in the first 15 min and then decreased rapidly to starting value with non-significant difference. The developed RSV nanocomposites attained satisfied pattern in FBS which suggest similar profile in in in vivo studies due absence  56 . Similar studies reported highlighting cytotoxicity of ZN against several types of cancers cells 44 .  Cellular uptake. Figure 5 exhibited that free RSV cellular uptake obtained 19.1 ± 1.3% and 31.4 ± 2.7% at 2 and 4 h of incubation, respectively. Significantly elevated cellular uptakes were observed with ZN-RSV NPs incubations, which reached 41.6 ± 23.1% and 62.3 ± 5.2% at after 2 and 4 h of incubation, respectively. The results indicated ameliorated cellular uptake significantly by ZN-RSV NPs compared to free RSV. This also confirmed the ability of ZN NPs to enhance HCT-116 cells uptake of RSV that may ameliorate pro-apoptotic profile. The obtained results concluded significant ameliorated RSV cellular uptake due to the developed formula. This confirmed the ability of ZN NPs to enhance HCT-116 cells uptake of RSV which also confirm the efficacy of ZN improved Enhanced Permeability and Retention (EPR) cellular uptake in tumor microenvironment. The cellular uptake of polymeric nanoparticles which possess < 200 nm mean diameter is described by an endocytotic action 57 . Also, positively charged nanoparticles generated a higher rate of membrane internalization in comparison with negatively charged ones due to enhanced adsorptive-mediated transcytosis catalyzed by electrostatic interaction yielded between positively charged structures of ZN and negatively charged cells membranes 58 .
Cell cycle progression. Rapid growth properties were exhibited from control untreated HCT-116 cells.
According to Fig. 6a, it obtained 52.06 ± 3.7% at the G0/G1 phase, 39.08 ± 2.9% at the S phase, 8.06 ± 0.4% at the G2-M phase, and 1.78 ± 0.6% at the pre-G1 phase. Other RSV, ZN and ZN-RSV NPs incubations displayed down the proliferation of HCT-116 cells, especially in the G0/G1 and S phases ( Fig. 6b-d). Regarding to accumulation of cells in the pre-G phase, it records 1.78 ± 0.6%, 23.14 ± 1.1%, 7.98 ± 0.5 and 25.72 ± 1.4% of control HCT-116 cells, RSV, ZN, and ZN-RSV NPs groups, respectively. RSV antiproliferative potential is correlated with apoptosis induction capability and G1 phase cell cycle arrest in colon cancer cells 59 . Also, the concluded pro-apoptotic activity was in harmony with the obtained RSV enhanced induced apoptosis in HCT-116 cells three-dimensional culture leading to formation of a luminal cavity by suppressing PDE4 activity 60 . Figure 6e exhibited the obtained changes in cell cycle phases graphically.
Annexin-V staining. In order to confirm the generated cell apoptotic death, the percentage of HCT-116 cells with positive annexin-V staining were estimated in the control, RSV, ZN and ZN-RSV NPs incubations ( Fig. 7a-d). ZN-RSV NPs distinctly increased the early, late, and total cell death in comparison with other incubations. A graphical explanation for cell death different types was explained in Fig. 7e. ZN-RSV NPs obtained the most functional activity in enhancing pre-G phase, which confirms apoptotic cell death. This indicated early and late apoptotic death, as well as total cell death. The obtained results confirmed the potential of RSV to induce apoptosis in colon cancer cells [61][62][63][64] . Similar RSV enhanced pro-apoptotic was obtained in HT29 colon cancer cell 65 . Also, these results support the utilization of ZN Nano-dispersed delivery systems for enhancing apoptotic and cellular uptake potential of piceatannol 55 and pterostilbene 56 . mRNA expression of apoptosis-related genes. Figure     www.nature.com/scientificreports/ RSV. These results confirmed the apoptotic role of RSV in cancer progression through modulation of microRNAs (miRNAs) which has not been studied 70 . Also, colon cancer progression was related to miRNAs that activating either as oncogenes or tumor-suppressor genes. In comparison to normal tissues, miRNAs have been dysregulated in colon cancer tissues 51 . NF-κB pathway is the fundamental contributor to colon tumors where RSV inverts NF-κB activation, which is responsible for inducing inflammatory cytokines 71 . The results of NF-κB expression showed folds change of 3.51, 3.8 and 4.96, respectively. ZN-RSV NPs displayed elevated anti-apoptotic effect in comparison with untreated positive control. ZN-RSV NPs attained 1.41-fold decreasing in NF-κB level in comparison with free RSV. As investigated by 72,73 NF-κB, as nuclear transcription factor involved in carcinogenesis, is implicated in cell proliferation, differentiation, apoptosis, tumorigenesis and cellular stress reactions. NF-κB activation might regulate several genes expression leading to cell survival. These results were in harmony with 74 where RSV was concluded to suppress NF-κB by blocking TNF-induced activation of NF-κB in a dose-and time-dependent manner. Fig. 9, ZN-RSV NPs obtained the largest green fluorescence intensity. ZN-RSV NPs obtained highest ROS activity (156.1 ± 9.4 Pg/106 million cells) which equal 1.27 upfolds of free RSV sample. This concluded that ZN-RSV NPs could induce oxidative stress in cancer cells by increasing ROS formation via oxidation of NADPH 75 . RSV demonstrated a protective potential regarding to ROS and nitric oxide synthesis 61 . ROS can generate subsequent toxic radicals like hydroxyl, hydrogen peroxide and superoxide which react to DNA and proteins on cellular level leading to DNA damage and lipid peroxidation 69,76 . The anti-peroxidative action of RSV was reported by 77 and correlated to capability of phenolic groups to regain lipid hydroperoxyl, hydroxyl and superoxide anions. Also, apoptosis induced by RSV in NCI-H460 non-small cell lung cancer cells and human colon cancer cells was contributed to ROS levels regulations in that trigger downstream signaling pathways as antioxidant or pro-oxidant 78,79 . eNOS activity assay. As reported in 51 eNOS is expressed in many cell lines as colon cell line HCT-116.

Reactive oxygen species assay (ROS). As displayed in
The response was explained as a response of tumor cells to the therapeutic agent. The results revealed increased levels of eNOS isoenzyme in ZN-RSV NPs sample in comparison with control and free RSV samples as exhibited in Fig. 10. There was increased significant production of eNOS in RSV and ZN-RSV NPs sample with obtained values of 10.72 and 12.58 ng/million cells, respectively. Zein base nano-particulates was a successful option for enhancing NO production induced by RSV in HCT-116 cells. This was in accordance with 80 where suppression of tumorigenicity and abrogation of cancer metastasis is related directly to direct NO production, which confirm apoptosis potential. Also, it has been concluded that RSV enhanced endothelial NO release through long and short-term effects where rapid effect comprises phosphorylation of eNOS by AMP-activated kinase, or extracellular signal regulated kinase 1 and 2, and deacetylation of eNOS by sirtuin1 81 . ZN-RSV NPs formulation. ZN-RSV NPs were prepared using nanoprecipitation method 40,42,55 . Three ZN-RSV NPs formulae (R1-R3) were prepared and varied according to the applied drug:polymer ratios. RSV and ZN were dissolved in 10 ml of 85% ethanol using of vortex (Velp scientifica, ZX3, Usmate, Italy) and ultrasonic probe (Vibra-Cell VCX750; Sonics and Materials Inc., Newtown, CT, USA). The ethanolic dispersion was flowed into deionized water and magnetically stirred at room temperature for 3 h at 2000 rpm to vaporize content of ethanol. The aqueous suspension was then centrifuged at 20,000×g speed then lyophilized using cryoprotectant of trehalose.

ZN-RSV NPs characterization.
Particle size analysis. Laser diffraction technique was utilized for particle size analysis of ZN-RSV NPs formulae (R1-R3). Using disposable cuvette, one milliliter of the sample was diluted in deionized water. The average particle size was then determined.
Encapsulation efficiency (EE). Ethanol solubilized samples were filtered through 0.22 µm filters, and analyzed for (EE) using reported high-performance liquid chromatography (HPLC) (Agilent 1200, Agilent Technologies, Santa Clara, CA, USA) equipped with a C18 column (5 μm, 4.6 mm × 250 mm), photodiode array detector (PDAD) at 306 nm (Waters, Milford, USA), adjusted column temperature at 35 °C, mixture of methanol and purified water (50:50 v/v) as mobile phase and 1 ml/min flow rate according to 82 . RSV EE was estimated according to the following equation: (1) EE (%) = amount of RSV in the nanocompsites amount of RSV initially added × 100.  The mediums were supplemented with fetal bovine serum to a final concentration of 10% and 20%. Culture plates and flasks (SPL Life Sciences, Korea) were subjected to addition of Penicillin (100 U/ml) and streptomycin (100 mg/ml) previously. Cells were maintained inside carbon dioxide incubator at 37 °C in a humidified atmosphere containing 5% CO 2 (Thermo Electron Corporation, Forma series II, 3141, USA) to keep the cells in a sub-confluent state.
Cytotoxicity assay. MTT assay was utilized to evaluate the antiproliferative activity using the kit of ABCAM, Cambridge, UK according to 83  Cell cycle progression analysis. This assay was carried out as described by 55,56 using the same reagents and kits.
Annexin-V assay. This assay was carried out as described by 55,56 using the same reagents and kits.
mRNA expression of apoptosis-related genes by quantitative real-time polymerase chain reaction (RT-PCR). This assay was carried out as reported by 55,56 using the same reagents and kits. Table 2 gives the primer sequences for caspase-3, miRNA125b, NF-κB and β-actin. Results were validated using the relative quantification ( ΔΔCT ) method. The genes expression was estimated in triplicates and runs mean was normalized with β-actin mean.
Reactive oxygen species assay (ROS). IC 50 concentrations of ZN-RSV NPs and equivalent concentrations of RSV and ZN were incubated in 96 well plates with adjusted cell density at 5 × 10 3 HCT-116 cells/well for period Endothelial nitric oxide synthase (eNOS) activity assay. Enzyme-linked immunosorbent assay kit (RayBio ® Human eNOS ELISA Kit, Norcross, GA, USA) was used to measure eNOS levels using 96-well plate. After adding of standards and samples into plates, biotinylated antihuman eNOS antibody is added where eNOS was present bounded to plate wells by immobilized antibody. HRP conjugated streptavidin is introduced to wells after washing. Second washing is carried out before the addition of TMB substrate solution. Then color was generated according to quantity of eNOS attached. Color was changed from blue to yellow after addition of stop solution. Finally, color intensity was measured at 450 nm.
Statistical analysis. Study data were displayed as means ± standard deviation (SD) of at least three independent runs. Comparative statistics between treatments was carried out by Student's t test or one-way analysis of variance followed by Tukey's test (criterion of significance was considered two-tailed p value less than 0.05). The utilized software was IBM SPSS software (Trial online version, SPSS Inc., Chicago, IL, USA).

Conclusions
The present study explored the ameliorating impact of nanoformulation on the molecular mechanism of RSV as anticancer molecule against Human CRC cells HCT-116. ZN-RSV NPs were fabricated with mean particle size of 137.6 ± 8.3 nm using the promising biomaterial of ZN. The developed ZN-RSV-NPs attained elevated EE%, 36 h sustained diffusion and colloidal stability. The enhanced cytotoxicity and cellular uptake, ameliorated pro-apoptotic potential supported by enhanced mRNA expression of caspase3, cleaved caspase-3, miRNA125b and NF-κB genes confirmed the enhanced anti-proliferative and pro-apoptotic potentials of ZN-RSV NPs. Biochemical examining exhibited increased generation of ROS and eNOS levels due to the ZN-RSV NPs formulation which displayed enhanced oxidant profile. The obtained results concluded therapeutical intensification of RSV antitumor potential against Human CRC cells HCT-116 due to the developed nano-delivery system.

Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.